PD-1 receptor outside the main paradigm: tumour-intrinsic role and clinical implications for checkpoint blockade.

Blocking the inhibitory receptor PD-1 on antitumour T lymphocytes is the main rationale underlying the clinical successes of cancer immunotherapies with checkpoint inhibitor (CI) antibodies (Abs). Besides this main paradigm, there is recent evidence of unconventional and "ectopic" signalling pathways of PD-1, found to be expressed not only by lymphocytes but also by peculiar subsets of cancer cells. Several groups reported on the tumour-intrinsic role of PD-1 in multiple settings, including melanoma, hepatocellular, thyroid, lung, pancreatic and colorectal cancer. Its functional activity appears intriguing but is not yet conclusively clarified. The initial studies are, in fact, supporting either a pro-tumourigenic role involved in chemoresistance and disease relapse or, oppositely, tumour-suppressive functions. The implications connected to the therapeutic administration of PD-1 blocking Abs are, of course, potentially relevant, respectively inferring an anti-tumour activity contrasting PD-1+ tumourigenic cells or a pro-tumoural effect by tackling PD-1 tumour suppressive signalling. The progressive exploration and consideration of this new paradigm of tumour-intrinsic PD-1 signalling may improve the interpretation of the observed clinical effects by anti-PD-1 Abs, likely resulting from multiple cumulative activities, and might provide important bases for dedicated clinical studies that take into account such composite roles of PD-1.

CD44v6 as innovative sarcoma target for CAR-redirected CIK cells.

Purpose of our study was to explore a new immunotherapy for high grade soft tissue sarcomas (STS) based on cytokine-induced killer cells (CIK) redirected with a chimeric antigen receptor (CAR) against the tumor-promoting antigen CD44v6. We aimed at generating bipotential killers, combining the CAR specificity with the intrinsic tumor-killing ability of CIK cells (CAR+.CIK). We set a patient-derived experimental platform. CAR+.CIK were generated by transduction of CIK precursors with a lentiviral vector encoding for anti-CD44v6-CAR. CAR+.CIK were characterized and assessed in vitro against multiple histotypes of patient-derived STS. The anti-sarcoma activity of CAR+.CIK was confirmed in a STS xenograft model. CD44v6 was expressed by 40% (11/27) of patient-derived STS. CAR+.CIK were efficiently expanded from patients (n = 12) and killed multiple histotypes of STS (including autologous targets, n = 4). The killing activity was significantly higher compared with unmodified CIK, especially at low effector/target (E/T) ratios: 98% vs 82% (E/T = 10:1) and 68% vs 26% (1:4), (p<0.0001). Specificity of tumor killing was confirmed by blocking with anti-CD44v6 antibody. CAR+.CIK produced higher amounts of IL6 and IFN-γ compared to control CIK. CAR+.CIK were highly active in mice bearing subcutaneous STS xenografts, with significant delay of tumor growth (p<0.0001) without toxicities. We report first evidence of CAR+.CIK's activity against high grade STS and propose CD44v6 as an innovative target in this setting. CIK are a valuable platform for the translation of CAR-based strategies to challenging field of solid tumors. Our findings support the exploration of CAR+.CIK in clinical trials against high grade STS.

The p85 regulatory subunit of PI3K mediates cAMP-PKA and insulin biological effects on MCF-7 cell growth and motility.

Recent studies have shown that hyperinsulinemia may increase the cancer risk. Moreover, many tumors demonstrate an increased activation of IR signaling pathways. Phosphatidylinositol 3-kinase (PI3K) is necessary for insulin action. In epithelial cells, which do not express GLUT4 and gluconeogenic enzymes, insulin-mediated PI3K activation regulates cell survival, growth, and motility. Although the involvement of the regulatory subunit of PI3K (p85α (PI3K)) in insulin signal transduction has been extensively studied, the function of its N-terminus remains elusive. It has been identified as a serine (S83) in the p85α (PI3K) that is phosphorylated by protein kinase A (PKA). To determine the molecular mechanism linking PKA to insulin-mediated PI3K activation, we used p85α (PI3K) mutated forms to prevent phosphorylation (p85A) or to mimic the phosphorylated residue (p85D). We demonstrated that phosphorylation of p85α (PI3K)S83 modulates the formation of the p85α (PI3K)/IRS-1 complex and its subcellular localization influencing the kinetics of the insulin signaling both on MAPK-ERK and AKT pathways. Furthermore, the p85α (PI3K)S83 phosphorylation plays a central role in the control of insulin-mediated cell proliferation, cell migration, and adhesion. This study highlights the p85α (PI3K)S83 role as a key regulator of cell proliferation and motility induced by insulin in MCF-7 cells breast cancer model.

Preparation of poly(methacrylic acid-g-poly(ethylene glycol)) nanospheres from methacrylic monomers for pharmaceutical applications.

Nanospheres of poly(methacrylic acid-grafted-poly(ethylene glycol)) were prepared by solution/precipitation polymerization. As colloidal drug delivery carriers, they present unique properties that render them promising candidates for oral protein delivery. The polymerization was carried out in water and the resulting suspension was freeze-dried. As with many colloidal systems, the freeze-dried suspension showed strong agglomeration after drying. The effects of preparation conditions on the particle size and redispersion were investigated using photon correlation spectroscopy. Furthermore, the ability of different types and concentrations of stabilizers (cryoprotectants and steric stabilizers) in preventing this phenomenon was addressed. Pluronics, block copolymers widely used as nonionic surfactants, were the most effective in stabilizing the particles during the freeze-drying process. Pluronic P123, however, increased significantly the particle size of the nanospheres. On the other hand, lyophilizates obtained in the presence of Pluronic F68 had good redispersion properties and no change in particle size was observed.

Physical properties of parabens and their mixtures: solubility in water, thermal behavior, and crystal structures.

The peculiar solubility behavior of propylparaben (propyl ester of 4-hydroxybenzoic acid) in aqueous solution, when tested separately and together with methyl-, ethyl-, and butyl-parabens, has been investigated in detail. The results clearly indicate that the decrease in solubility (approximately 50% compared to the solubility value of propylparaben alone) is typical of those mixtures containing also ethylparaben, as demonstrated by solubility experiments on binary, ternary, and quaternary mixtures of the parabens. Phase diagrams of all the six binaries show that propylparaben and ethylparaben are the only pair that form almost ideal solid solutions near the melting temperatures. Moreover, phase-solubility analysis shows that propylparaben and ethylparaben, at room temperature, can also form solid solutions whose solubility is related to the composition of the solid phase at equilibrium. To achieve an independent confirmation of the possible solid solution formation that supports the above interpretation of the solubility behavior, the crystal structures of the four parabens have been examined and isostructurality has been found to exist only between ethylparaben and propylparaben. Powder X-ray diffraction has also been performed on ethylparaben, propylparaben, and their solid solutions obtained by recrystallization from water. The progressive shift of distinctive diffraction peaks with phase composition clearly indicates that propylparaben and ethylparaben form substitutional solid solutions. The small value (<1) of the disruption index provides thermodynamic support for substitutional solid solutions based on isostructural crystals.